BHLH47 Antibody

What is the basic structure and function of BHLH47 protein?

BHLH47 belongs to the basic helix-loop-helix (bHLH) family of transcription factors. Like other bHLH proteins, it contains a conserved domain of approximately 60 amino acids with two functionally distinct regions: a basic DNA-binding region at the N-terminal end and a helix-loop-helix (HLH) dimerization domain at the C-terminal end . The basic region consists of about 15 amino acids with numerous basic residues that interact directly with DNA, while the HLH region contains primarily hydrophobic residues forming two amphipathic α-helices separated by a variable loop region .

The functional mechanism involves the interaction between HLH regions of two separate polypeptides, leading to homodimer or heterodimer formation. Each partner's basic region then binds to half of the DNA recognition sequence . BHLH47, like other members of this family, recognizes a consensus hexanucleotide sequence known as the E-box (5′-CANNTG-3′), with potential specificity for particular types of E-boxes depending on the central two nucleotides and flanking sequences .

How do I determine the appropriate BHLH47 antibody concentration for Western blotting?

When establishing optimal antibody dilutions for BHLH47 detection via Western blotting, a systematic titration approach is essential. Begin with the manufacturer's recommended range, typically between 1:500 and 1:2000 for primary antibodies. Prepare a dilution series (e.g., 1:500, 1:1000, 1:2000) and test them in parallel under identical conditions.

The optimal concentration will provide strong specific signal (your BHLH47 band at the expected molecular weight of approximately 25-27 kDa, similar to other bHLH proteins like Twist-1) with minimal background . If detecting recombinant BHLH47, reference the specific construct information - for instance, full-length human Twist-1 spans Met1-His202 .

When optimizing:

• Always include positive and negative controls

• Maintain consistent protein loading (20-50 μg total protein)

• Document exposure times precisely

• Consider signal-to-noise ratio rather than absolute signal intensity

• Validate specificity with knockout/knockdown samples if available

As noted in antibody documentation for similar bHLH proteins: "Optimal dilutions should be determined by each laboratory for each application" .

What are the recommended methods for analyzing BHLH47 dimerization partners in cellular contexts?

Analyzing BHLH47 dimerization requires multiple complementary approaches to identify and validate interaction partners. Based on established protocols for other bHLH proteins:

Co-immunoprecipitation (Co-IP):
This remains the gold standard for detecting native protein interactions. Using a validated BHLH47 antibody, precipitate the protein complex from cell lysates, then probe for potential partners via Western blotting. For novel interactions, follow with mass spectrometry identification.

Bimolecular Fluorescence Complementation (BiFC):
This technique visualizes protein interactions in living cells by fusing potential interaction partners to complementary fragments of a fluorescent protein (e.g., Venus). When BHLH47 and its partner interact, the fragments reconstitute a functional fluorophore, generating visible fluorescence .

Fluorescence Resonance Energy Transfer (FRET):
Tag BHLH47 and potential partners with appropriate donor-acceptor fluorophore pairs (e.g., CFP/YFP). Interaction places these fluorophores in proximity, enabling energy transfer that can be quantified by microscopy or flow cytometry.

Yeast Two-Hybrid (Y2H) Screening:
While not suitable for all interactions, Y2H can identify novel partners from cDNA libraries. Validate hits with the methods above in mammalian cells.

Cross-linking studies:
UV cross-linking has been successfully applied to bHLH proteins like E47, allowing researchers to "freeze" dimerization states for subsequent analysis .

When investigating specific heterodimer formation, use "forced heterodimer" approaches similar to those employed in E47 studies, which revealed that in B cells, E2A proteins primarily form homodimers rather than binding B-cell-restricted partners .

How can I detect and quantify BHLH47 binding to specific DNA sequences?

Several complementary methods allow for robust analysis of BHLH47 DNA-binding specificity and affinity:

Chromatin Immunoprecipitation (ChIP):
ChIP using a high-quality BHLH47-specific antibody remains the standard for identifying genomic binding sites in vivo. For genome-wide binding profiles, combine with next-generation sequencing (ChIP-seq). Careful optimization of crosslinking conditions and sonication parameters is essential for reproducible results.

Electrophoretic Mobility Shift Assay (EMSA):
For in vitro binding analysis, EMSA provides direct visualization of protein-DNA interactions. Design oligonucleotides containing putative E-box sequences (5'-CANNTG-3') with several flanking nucleotides, as these influence binding specificity . Include competition assays with unlabeled probes to confirm specificity.

DNA-Protein Binding Microarrays:
These high-throughput platforms evaluate binding to thousands of DNA sequences simultaneously, allowing comprehensive motif determination. Similar approaches have established detailed binding preferences for multiple bHLH factors, revealing quantitative differences in affinity toward various E-box configurations .

High-Throughput Systematic Evolution of Ligands by Exponential Enrichment (HT-SELEX):
This method identifies preferred binding sequences from large random oligonucleotide pools and has been successfully applied to bHLH factors .

Reporter Gene Assays:
To assess functional consequences of binding, use luciferase reporters with wild-type and mutated E-box sequences. This validates the biological relevance of binding events observed through other methods.

When analyzing results, remember that bHLH factors often recognize half-sites (CAN) on opposing DNA strands, making the orientation of these half-sites informative of dimer configuration .

How does post-translational modification affect BHLH47 antibody detection and protein function?

Post-translational modifications (PTMs) significantly impact both antibody recognition and biological function of bHLH proteins, including BHLH47. Understanding these effects is crucial for accurate experimental interpretation.

Effects on Antibody Detection:
Multiple PTMs can affect epitope recognition by antibodies. The most common PTMs affecting bHLH proteins include:

Functional Implications:
PTMs regulate multiple aspects of bHLH protein function:

• DNA binding affinity and specificity

• Dimerization partner preferences

• Protein stability and turnover

• Subcellular localization

When investigating BHLH47 function, consider using phosphatase inhibitors during sample preparation to preserve physiological modification states. For functional studies, compare wild-type protein with phosphomimetic (S/T→D/E) or non-phosphorylatable (S/T→A) mutants to elucidate the role of specific modifications.

CDK-mediated phosphorylation has been documented to regulate activity of several bHLH factors and may similarly control BHLH47 function in cell cycle-dependent contexts .

What are the best approaches for real-time imaging of BHLH47 dynamics in live cells?

Real-time imaging of BHLH47 offers invaluable insights into its temporal dynamics and spatial regulation. Based on successful approaches with other bHLH factors, several strategies are recommended:

Fluorescent Fusion Proteins:
Creating knock-in or BAC transgenic systems expressing fluorescent protein fusions (e.g., Venus-BHLH47 or mCherry-BHLH47) provides the most physiologically relevant system for visualization . When designing these constructs:

• Place the fluorescent tag at either N- or C-terminus, avoiding disruption of the bHLH domain

• Validate that fusion proteins retain wild-type localization and function

• Consider using destabilized fluorescent proteins for capturing rapid dynamics

Live-Cell Confocal Microscopy:
For high-resolution spatial information, confocal microscopy with environmental control (temperature, CO₂) enables tracking of BHLH47 translocation between cytoplasm and nucleus in response to signaling events.

Fluorescence Recovery After Photobleaching (FRAP):
FRAP analysis measures the mobility and binding dynamics of BHLH47 within different cellular compartments, revealing information about its association with chromatin or other nuclear structures.

Bioluminescence Resonance Energy Transfer (BRET):
For detecting protein-protein interactions in real-time, BRET offers advantages over FRET by eliminating the need for external illumination, reducing phototoxicity during long-term imaging .

Previous studies with bHLH transcription factors have revealed oscillatory expression patterns in neural stem cells that would have been impossible to detect without live imaging techniques . Similar dynamic regulation might exist for BHLH47, particularly in developmental or differentiation contexts.

When designing experiments, acquire images at appropriate intervals based on the expected dynamics (minutes to hours) and minimize laser power to reduce photobleaching and phototoxicity.

How do I validate BHLH47 antibody specificity and minimize cross-reactivity with other bHLH family members?

Given the structural conservation among the 150+ members of the bHLH family, rigorous validation of antibody specificity is paramount. The following comprehensive validation strategy ensures reliable results:

Genetic Controls:
The gold standard for antibody validation is testing with genetic knockout/knockdown systems:

• CRISPR/Cas9-mediated BHLH47 knockout cells

• siRNA or shRNA-mediated knockdown (with 70-90% reduction in target expression)

• Overexpression systems using tagged BHLH47 constructs

Peptide Competition Assays:
Pre-incubate your antibody with the immunizing peptide before applying to your sample. Specific signals should be abolished or significantly reduced.

Multi-Antibody Concordance:
Use at least two antibodies targeting different epitopes of BHLH47. Concordant results across antibodies significantly increase confidence in specificity.

Cross-Reactivity Testing:
Express multiple bHLH family members in a controlled system and test your antibody against each. Focus particularly on closely related family members that share higher sequence homology in the epitope region. The classification system described by Heim et al. (2003) can guide selection of the most relevant potential cross-reactants .

Epitope Mapping:
Understand precisely which region of BHLH47 your antibody recognizes. Antibodies targeting the highly conserved bHLH domain (particularly residues involved in DNA binding or dimerization) have higher cross-reactivity risk compared to those targeting divergent N- or C-terminal regions .

Application-Specific Validation:
An antibody working well in Western blotting may fail in immunoprecipitation or immunofluorescence. Validate separately for each application using appropriate controls.

Remember that the bHLH consensus motif shows conservation at specific positions (Table 1 in reference ), and antibodies recognizing these conserved residues may demonstrate cross-reactivity.

What are the key differences in experimental design when studying BHLH47 homodimers versus heterodimers?

The experimental approach differs substantially when investigating homodimeric versus heterodimeric forms of BHLH47, requiring careful consideration of the following factors:

Sample Preparation:
For homodimer studies, expression of BHLH47 alone is sufficient. For heterodimer analysis, both partners must be present in appropriate stoichiometric ratios. In overexpression systems, use equimolar amounts of expression vectors or consider bicistronic constructs to ensure consistent co-expression.

Protein Interaction Analysis:
When studying homodimers, standard immunoprecipitation with a single antibody is adequate. For heterodimers, reciprocal co-immunoprecipitation is essential - pull down with anti-BHLH47 and probe for partner, then repeat by pulling down partner and probing for BHLH47.

DNA Binding Studies:
Homodimers and heterodimers recognize distinct E-box variants. Design oligonucleotides containing different E-box sequences (CACGTG, CAGCTG, CATGTG, etc.) for EMSA studies. Heterodimers often show binding to composite elements not recognized by either homodimer alone .

Functional Readouts:
Evaluate transcriptional effects using reporters with homodimer-preferred versus heterodimer-preferred E-box variants. Mutation analysis of specific half-sites can distinguish between contributions of each partner in heterodimeric complexes.

Structural Analysis:
For advanced studies, techniques differ significantly:

• For homodimers, symmetrical crosslinking approaches are effective

• For heterodimers, asymmetric crosslinking or "forced heterodimers" (similar to those used in E47 studies ) are required

Dimerization Mutants:
Create specific mutations in the HLH region that selectively disrupt homo- or heterodimerization. The conserved HLH residues responsible for dimerization have been mapped through crystallographic studies of other bHLH proteins and can guide mutation design .

Remember that some bHLH proteins like E47 can form both homo- and heterodimers, with distinct functional outcomes in different cellular contexts . The relative abundance of potential partner proteins significantly influences dimer formation in vivo.

How can single-cell analysis techniques be applied to study BHLH47 expression heterogeneity in tissue samples?

Single-cell approaches offer unprecedented insights into the heterogeneous expression and function of BHLH47 within complex tissues. Implementing these techniques requires careful consideration of several methodological aspects:

Single-Cell RNA Sequencing (scRNA-seq):
For transcriptional profiling, several platforms (10x Genomics, Drop-seq, Smart-seq2) can detect BHLH47 mRNA in individual cells. When designing scRNA-seq experiments:

• Ensure adequate sequencing depth (minimum 50,000 reads/cell) for reliable detection of transcription factors, which typically have lower expression than housekeeping genes

• Validate key findings with single-molecule FISH for spatial context

• Use computational deconvolution to identify co-expression patterns with potential dimerization partners

Single-Cell Protein Analysis:
While challenging for transcription factors, these approaches provide protein-level data:

• Mass cytometry (CyTOF) with metal-conjugated BHLH47 antibodies

• Single-cell Western blotting

• Microfluidic antibody capture assays

Spatial Transcriptomics:
These methods preserve tissue context while providing single-cell resolution:

• MERFISH (Multiplexed Error-Robust FISH)

• Slide-seq

• Visium Spatial Gene Expression (10x Genomics)

Integrated Single-Cell Analysis:
Combining modalities provides the most comprehensive picture:

• CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) - simultaneously measures BHLH47 mRNA and protein

• scATAC-seq paired with scRNA-seq reveals chromatin accessibility at BHLH47 binding sites

Studies of other bHLH factors have revealed striking cell-to-cell variability in expression levels within seemingly homogeneous populations. For instance, immunostaining of neural stem cells showed that while Sox2 expression remained relatively constant, Hes1, Ascl1, and Olig2 levels varied considerably between cells . Similar heterogeneity may exist for BHLH47, potentially reflecting different functional states or cell cycle positions.

What are the emerging techniques for studying temporal dynamics of BHLH47 binding to chromatin?

Recent technological advances have transformed our ability to capture the dynamic nature of transcription factor-chromatin interactions. For studying BHLH47 binding dynamics, consider these cutting-edge approaches:

Time-Resolved ChIP-seq:
Standard ChIP provides a static snapshot, but time-resolved approaches reveal binding kinetics:

• Calibrated ChIP-seq with spike-in controls for quantitative comparisons across timepoints

• ChIP-BIT-seq (Barcoded, Inducible, and Time-resolved ChIP-seq) for automated temporal profiling

• CUT&RUN or CUT&Tag methods for higher sensitivity with lower cell numbers

Live-Cell Chromatin Imaging:
Direct visualization of BHLH47-chromatin interactions:

• CRISPR-dCas9 systems with fluorescently tagged BHLH47 to monitor binding at specific genomic loci

• Single-particle tracking to measure residence times on chromatin

• lattice light-sheet microscopy for 3D tracking with minimal phototoxicity

Nascent Transcription Assays:
Connect dynamic binding to functional outputs:

• PRO-seq or GRO-seq to measure immediate transcriptional consequences of binding

• TT-seq (Transient Transcriptome sequencing) for short-lived RNAs

• SLAM-seq for newly synthesized RNA detection

Genome Architecture Mapping:
Map 3D interactions between BHLH47 binding sites:

• HiChIP or PLAC-seq to selectively enrich for chromatin interactions involving BHLH47

• Micro-C for nucleosome-resolution contact maps

Real-time imaging studies of bHLH transcription factors in neural stem cells have revealed oscillatory expression patterns that drive cell fate decisions . These findings suggest that temporal dynamics of BHLH47 may be equally important for its biological function. When designing experiments to capture these dynamics, consider appropriate sampling intervals (minutes to hours) based on the biological process under investigation.

The choice of synchronization method (if needed) should be carefully evaluated, as it may introduce artifacts. Whenever possible, use live-cell approaches that don't require cell population synchronization.